Site-directed Editing Of NtMYB4a Gene Using CRISPR/Cas9 Technology And Its Molecular Mechanism In Response To Low Temperature Stress

Tobacco(Nicotiana tabacum)is one of the economic crops widely planted.It is a thermophilic crop and is very sensitive to temperature changes.Low temperature will cause the tobacco seedlings to grow slowly,the leaves are narrow and long,the number of leaves is reduced,and the early blooming phenomenon occurs,thereby reducing the yield and quality of tobacco leaves.MYB transcription factor participates in the regulation of a variety of physiological and biochemical processes in plants,and plays an important role in resisting low temperature injury and other adverse environments.There are more than 300 MYB transcription factors in the tobacco genome,but there are few reports on their functions.The previous research of the research group found that a R2R3-MYB transcription factor NtMYB4 a has a clear response to low temperature stress.In order to further explore the function of this gene under low temperature stress and its regulatory mechanism,In this study,a CRISPR/Cas9 knockout vector of NtMYB4 a was constructed and introduced into tobacco plants by agrobacterium-mediated method,and transgenic plants will be successfully obtained.The transgenic plants and wild-type tobacco plants were treated with low temperature stress,the expression of NtMYB4 a and stress response genes and the physiological function changes of the plants were analyzed,and the physiological and molecular mechanisms of NtMYB4 a involved in low temperature stress were preliminarily analyzed to provide genetic resources and mutants for tobacco stress resistance engineering.Provide a scientific basis for the improvement of tobacco resistance to low temperature characteristics.The main research results are as follows:(1)Two targets Y1 and B1 were selected at exon 1 and exon 3 of NtMYB4 a.The double target knockout vector of CRISPR/cas9 editing system was constructed and introduced into tobacco by agrobacterium mediated method,and 119 transformants were obtained.29 positive plants were screened by PCR,and the positive rate was24.37%.The PCR products of 29 positive tobacco strains were sequenced,and 15 of them had different degrees of mutation at the target and non target sites,the mutation rate was 51.72%.(2)There are 4 mutation types in 15 mutant strains.There are 4 strains of mutation type I,and base deletions,substitutions or insertions occur respectively at the target and non-target sites,causing missense,synonymous or frameshift mutations of amino acids;1 strain of mutation type II occurs at the target site Base substitutions cause synonymous and missense mutations and produce stop codons;one strain of mutation type III has base substitutions,insertions and deletions at target and non-target sites,causing missense and frameshift mutations of amino acids;There are9 strains of mutation type IV.Base deletions and substitutions occur at the target and non-target sites,causing frameshift mutations and missense mutations of amino acids,and the stop code TAA is generated,which makes protein translation stop prematurely.(3)The expression of NtMYB4 a in the flowers,stems and leaves of different mutant tobacco plants are all down-regulated,but the down-regulated expression of NtMYB4 a varies depending on the type and tissues and organs.The expression of NtMYB4 a was the lowest in the flower and middle leaf of mutant K10,the lowest in the stem of K17 and the lowest in the upper leaf of K16.The plant height of the mutant tobacco plants is shorter than that of the wild type,and the flower color is lighter than that of the wild type.Further analysis found that the anthocyanin content of the mutant tobacco plants was lower than that of the wild type,and the expression levels of the anthocyanin synthesis-related enzyme genes PAL,C4 H,4CL,ANS and DFR were all down-regulated.The leaf color of the mutant K17 at the mature stage was darker than that of the wild type,with obvious wrinkles between the veins,and the mesophyll cells of the palisade tissue contained more chloroplasts.The results show that NtMYB4 a is involved in the synthesis of tobacco anthocyanins and the growth and development of tobacco plants.(4)Under low temperature stress,the wilting degree of different mutant types of tobacco plants is more serious than that of wild type tobacco plants,with lower proline content and higher MDA content,indicating that the mutation of NtMYB4 a leads to reduced resistance to low temperature of tobacco plants.Under low temperature stress,the content of PAL enzyme,lignin,anthocyanin,SOD enzyme and MDA in the mutant strain was higher than that of the wild type,and the content of proline and CAT enzyme was lower than that of the wild type.Low temperature stress increased the expression of NtMYB4 a in wild-type and mutant strains and the expression of lignin and anthocyanin synthesis-related enzyme genes PAL,C4 H,4CL,ANS,DFR and HCT,but in mutant strains NtMYB4 a,PAL,C4 H,4CL,HCT the up-regulated expression of is higher than that of wild-type.Therefore,it is suggested that low temperature induces the up-regulation of NtMYB4 a expression,and regulates the expression of PAL,C4 H,4CL,ANS,DFR and HCT to promote the accumulation of lignin and anthocyanin to resist low temperature stress.(5)Under low temperature stress,the expression of proline,chlorophyll,lignin,anthocyanin content,PAL enzyme activity,SOD enzyme activity,MDA and NtMYB4 a,PAL,C4 H,4CL,ANS,DFR,HCT of NtMYB4 a mutant strains K17 and K16 increased.However,the proline,chlorophyll,lignin,anthocyanin content,POD,CAT enzyme activity,PAL and ASN expression levels in K17 are all higher than those in K16,the content of MDA and chlorogenic acid and the expression of C4 H,4CL and HCT are lower than K16,It shows that the physiological responses of tobacco plants of different mutation types are inconsistent under low temperature stress.